The retinal pigmented epithelium (RPE) is a layer of cells in the eye. The RPE is overlaid by the sensory retina cells which perceive light and transmit visual information to the optic nerve. Underlying the RPE is the choroid tissue, a vascularized region which supplies the overlying cells of the eye with water, nutrients and other compounds. The RPE plays many critical roles in maintaining vision including isolating the tissues of the eye from the general circulatory system, maintaining the proper ionic environment, processing discarded outer photoreceptor elements from the photoreceptor cells of the neural retina, and protecting the retina from excess light. RPE cells form a flat mosaic of hexagonal cells tightly bound at their junctions.
Various conditions may result in damage and dysfunction of the RPE cells. For example, in some forms of retinitis pigmentosa, RPE cells exhibit abnormalities and dysfunction that affect vision. Another example is age-related macular degeneration (AMD), a disease that gradually diminishes vision in the macula, or central region of the eye. AMD is a leading cause of vision loss in persons 60 years of age and older. It is estimated that in the United States 30% of people over age 75 suffer from some form of AMD. There are few therapies available for AMD and none that effectively cure or reverse the condition. In some forms of AMD, deposits of cellular debris (drusen) form between the RPE and the underlying nourishing choroid, leading to death and dysfunction of the RPE cells. Choroidal neovascularization (CNV) is an AMD subtype characterized by abnormal blood vessel proliferation of the choroidal tissue and the resultant loss of vision resulting from damage to the overlying retinal cells. Geographic atrophy is another form of AMD characterized by atrophy of the retinal pigmented epithelial cells and the resultant death of the overlying retinal cells.
It has been demonstrated, both in animal tests and in human trials, that transplantation of healthy RPE cells to damaged or destroyed regions of the retina can aid in restoring vision (da Cruz et al., RPE transplantation and its role in retinal disease, Progress in Retinal and Eye Research 26:598-635 (2007)). For example, in patients with CNV, surgery to remove the heavily vascularized tissue followed by transplant of RPE cells from the patients' own eyes was shown to restore vision (Chen, et al., Long-term visual and microperimetry outcomes following autologous retinal pigment epithelium choroid graft for neovascular age-related macular degeneration, Clinical and Experimental Ophthamology 37:275-285 (2009)).
To maximize therapeutic potential, it would be advantageous to obtain large supplies of immune-compatible RPE cells for transplant purposes. RPE cells derived from stem cells and induced pluripotent cells present such a potential source of abundant RPE tissues for transplant. For example, RPE cells derived from both human embryonic and induced pluripotent stem cells have been created and transplanted in rats and were shown to be functional (Carr et al., Protective Effects of Human iPS-Derived Retinal Pigment Epithelium Cell Transplantation in the Retinal Dystrophic Rat, PloS One 4(12):e8152 (2009)). RPE cells from human embryonic stem cells are currently in clinical trials for the treatment of both AMD and Stargardt's macular dystrophy.
The derivation of RPE cells from pluripotent cells has been demonstrated previously, for example, as described in Buchholz et al., Derivation of Functional Retinal Pigmented Epithelium from Induced Pluripotent Stem Cells, Stem Cells 27:2427-2434 (2009), and in Can et al., Molecular Characterization and functional analysis of phagocytosis by human embryonic stem cell-derived RPE cells using a novel human retinal assay, Mol Vis 15:283-295 (2009). However, the known protocols for deriving RPE from pluripotent cells are inefficient, typically giving rise to 1-5% differentiated RPE cells. Only one group has reported substantially higher yields, reporting a yield of 33% differentiated RPE cells (Idelson, M., R. Alper, et al. (2009). “Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells.” Cell Stem Cell 5(4): 396-408). In addition to the low yields of the prior art methods, these methods require many months for the production of functional RPE cells in usable quantities. Accordingly, there is a need in the art for facile, high yielding and highly consistent methods of creating viable RPE cells from pluripotent cells.